Attenuation of DNA base oxidation in post-surgery colorectal stage III patients at subsequent follow-ups.

DNA damage caused by oxidative reactions plays a crucial role in the pathogenesis of colorectal cancer (CRC). In a previous cross-sectional study, CRC patients diagnosed with regional disease (stage III) exhibited a higher level of DNA base oxidation in peripheral blood mononuclear cells (PBMCs) 2-9 months post-surgery compared to those with localized disease (stage I-II). To further explore this observation over time, the present study aimed to investigate DNA base oxidation in CRC patients with localized versus regional disease 6 and 12 months after the initial measurements. The present study included patients enrolled in the randomized controlled trial Norwegian Dietary Guidelines and Colorectal Cancer Survival (CRC-NORDIET). The standard comet assay, modified with the lesion-specific enzyme formamidopyrimidine DNA glycosylase (Fpg), was applied to measure DNA base oxidation in PBMCs at the 6- and 12-month follow-ups. Of the 255 patients assessed at baseline, 156 were included at the 6-month follow-up, with 89 of these patients included in the 12-month follow-up. In contrast to our observation at baseline, there were no significant differences in the levels of DNA base oxidation between patients diagnosed with localized disease and those with regional involvement at the 6- and 12-month follow-up visits (P = 0.81 and P = 0.09, respectively). Patients with stage III disease exhibited a significant decrease in the levels of DNA base oxidation from baseline to 6 months (P < 0.01) and baseline to 12 months (P = 0.03), but no significant difference from 6 to 12 months (P = 0.80). In conclusion, the initially elevated levels of DNA base oxidation in PBMCs, observed 2-9 months post-surgery in patients diagnosed with regional disease (stage III), subsequently decreased to levels comparable to patients with localized disease (stage I-II) at the 6- and 12-month follow-ups.

Lack of cytotoxic and genotoxic effects of mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium despite cellular uptake in cancerous and noncancerous lung cells.

Cytotoxic and genotoxic effects of novel mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium (Mg0.1-γ-Fe2O3(mPEG-silane)0.5) have been investigated on human adenocarcinomic alveolar basal epithelial (A549) and human normal bronchial epithelial (BEAS-2B) cells. In the studies several molecular and cellular targets addressing to cell membrane, cytoplasm organelles and nucleus components were served as toxicological endpoints. The as-synthesized nanoparticles were found to be stable in the cell culture media and were examined for different concentration and exposure times. No cytotoxicity of the tested nanoparticles was found although these nanoparticles slightly increased reactive oxygen species in both cell types studied. Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles did not produce any DNA strand breaks and oxidative DNA damages in A549 and BEAS-2B cells. Different concentration of Mg0.1-γ-Fe2O3(mPEG-silane)0.5 nanoparticles and different incubation time did not affect cell migration. The lung cancer cells' uptake of the nanoparticles was more effective than in normal lung cells. Altogether, the results evidence that mPEG-silane coated iron(III) oxide nanoparticles doped with magnesium do not elucidate any deleterious effects on human normal and cancerous lung cells despite cellular uptake of these nanoparticles. Therefore, it seems reasonable to conclude that these novel biocompatible nanoparticles are promising candidates for further development towards medical applications.

Effect of a personalized intensive dietary intervention on base excision repair (BER) in colorectal cancer patients: Results from a randomized controlled trial.

DNA repair is essential to maintain genomic integrity and may affect colorectal cancer (CRC) patients' risk of secondary cancers, treatment efficiency, and susceptibility to various comorbidities. Bioactive compounds identified in plant foods have the potential to modulate DNA repair mechanisms, but there is limited evidence of how dietary factors may affect DNA repair activity in CRC patients in remission after surgery. The aim of this study was to investigate the effect of a 6-month personalized intensive dietary intervention on DNA repair activity in post-surgery CRC patients (stage I-III). The present study included patients from the randomized controlled trial CRC-NORDIET, enrolled 2-9 months after surgery. The intervention group received an intensive dietary intervention emphasizing a prudent diet with specific plant-based foods suggested to dampen inflammation and oxidative stress, while the control group received only standard care advice. The comet-based in vitro repair assay was applied to assess DNA repair activity, specifically base excision repair (BER), in peripheral blood mononuclear cells (PBMCs). Statistical analyses were conducted using gamma generalized linear mixed models (Gamma GLMM). A total of 138 CRC patients were included, 72 from the intervention group and 66 from the control group. The BER activity in the intervention group did not change significantly compared to the control group. Our findings revealed a substantial range in both inter- and intra-individual levels of BER. In conclusion, the results do not support an effect of dietary intervention on BER activity in post-surgery CRC patients during a 6-month intervention period.

An ecotoxicological assessment of a strigolactone mimic used as the active ingredient in a plant biostimulant formulation.

A risk assessment on the aquatic toxicity of the plant biostimulant strigolactone mimic (2-(4-methyl-5-oxo-2,5-dihydro-furan-2-yloxy)-benzo[de]isoquinoline-1,3-dione (SL-6) was performed using a suite of standardised bioassays representing different trophic groups and acute and chronic endpoints. In freshwater, three trophic groups of algae, crustacea and fish were used. Whilst in seawater, algae (unicellular and macroalgae), Crustacea and Mollusca were employed. In addition, the genotoxicity of SL-6 was determined with the comet assessment performed on unicellular marine algae, oysters, and fish embryos. This was the first time ecotoxicity tests have been performed on SL-6. In freshwater, the lowest LOEC was measured in the unicellular algae at 0.31 mg/L SL-6. Although, similar LOEC values were found for embryo malformations and impacts on hatching rate in zebrafish (LOEC 0.31-0.33 mg/L). Consistent malformations of pericardial and yolk sac oedemas were identified in the zebrafish embryos at 0.31 mg/L. In marine species, the lowest LOEC was found for both Tisbe battagliai mortality and microalgae growth at an SL-6 concentration of 1.0 mg/L. Significant genotoxicity was observed above control levels at 0.0031 mg/L SL-6 in the unicellular algae and 0.001 mg/L SL-6 in the oyster and zebrafish larvae. When applying the simple risk assessment, based on the lowest NOECs and appropriate assessment factors, the calculated predicted no effect concentration (PNEC), for the ecotoxicity and the genotoxicity tests were 1.0 µg/L and 0.01 µg/L respectively.

DNA base oxidation in relation to TNM stages and chemotherapy treatment in colorectal cancer patients 2-9 months post-surgery.

Accumulation of DNA damage is a critical feature of genomic instability, which is a hallmark of various cancers. The enzyme-modified comet assay is a recognized method to detect specific DNA lesions at the level of individual cells. In this cross-sectional investigation, we explore possible links between clinicopathological and treatment related factors, nutritional status, physical activity and function, and DNA damage in a cohort of colorectal cancer (CRC) patients with non-metastatic disease. Levels of DNA damage in peripheral mononuclear blood cells (PBMCs) assessed 2-9 months post-surgery, were compared across tumour stage (localized (stage I-II) vs. regional (stage III) disease), localization (colon vs. rectosigmoid/rectum cancer), and adjuvant chemotherapy usage, with the last dosage administrated 2-191 days prior to sampling. Associations between DNA damage and indicators of nutritional status, physical activity and function were also explored. In PBMCs, DNA base oxidation was higher in patients diagnosed with regional compared with localized tumours (P = 0.03), but no difference was seen for DNA strand breaks (P > 0.05). Number of days since last chemotherapy dosage was negatively associated with DNA base oxidation (P < 0.01), and patients recently receiving chemotherapy (<15 days before blood collection) had higher levels of DNA base oxidation than those not receiving chemotherapy (P = 0.03). In the chemotherapy group, higher fat mass (in kg and %) as well as lower physical activity were associated with greater DNA base oxidation (P < 0.05). In conclusion, DNA base oxidation measured with the enzyme-modified comet assay varies according to tumour and lifestyle related factors in CRC patients treated for non-metastatic disease.

DNA strand break levels in cryopreserved mononuclear blood cell lines measured by the alkaline comet assay: results from the hCOMET ring trial.

The comet assay is widely used in biomonitoring studies for the analysis of DNA damage in leukocytes and peripheral blood mononuclear cells. Rather than processing blood samples directly, it can be desirable to cryopreserve whole blood or isolated cells for later analysis by the comet assay. However, this creates concern about artificial accumulation of DNA damage during cryopreservation. In this study, 10 laboratories used standardized cryopreservation and thawing procedures of monocytic (THP-1) or lymphocytic (TK6) cells. Samples were cryopreserved in small aliquots in 50% foetal bovine serum, 40% cell culture medium, and 10% dimethyl sulphoxide. Subsequently, cryopreserved samples were analysed by the standard comet assay on three occasions over a 3-year period. Levels of DNA strand breaks in THP-1 cells were increased (four laboratories), unaltered (four laboratories), or decreased (two laboratories) by long-term storage. Pooled analysis indicates only a modest positive association between storage time and levels of DNA strand breaks in THP-1 cells (0.37% Tail DNA per year, 95% confidence interval: -0.05, 0.78). In contrast, DNA strand break levels were not increased by cryopreservation in TK6 cells. There was inter-laboratory variation in levels of DNA strand breaks in THP-1 cells (SD = 3.7% Tail DNA) and TK6 reference sample cells (SD = 9.4% Tail DNA), whereas the intra-laboratory residual variation was substantially smaller (i.e. SD = 0.4%-2.2% Tail DNA in laboratories with the smallest and largest variation). In conclusion, the study shows that accumulation of DNA strand breaks in cryopreserved mononuclear blood cell lines is not a matter of concern.

Long-term cryopreservation of potassium bromate positive assay controls for measurement of oxidatively damaged DNA by the Fpg-modified comet assay: results from the hCOMET ring trial.

The formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay is widely used for the measurement of oxidatively generated damage to DNA. However, there has not been a recommended long-term positive control for this version of the comet assay. We have investigated potassium bromate as a positive control for the Fpg-modified comet assay because it generates many Fpg-sensitive sites with a little concurrent generation of DNA strand breaks. Eight laboratories used the same procedure for the treatment of monocytic THP-1 cells with potassium bromate (0, 0.5, 1.5, and 4.5 mM) and subsequent cryopreservation in a freezing medium consisting of 50% foetal bovine serum, 40% RPMI-1640 medium, and 10% dimethyl sulphoxide. The samples were analysed by the Fpg-modified comet assay three times over a 3-year period. All laboratories obtained a positive concentration-response relationship in cryopreserved samples (linear regression coefficients ranging from 0.79 to 0.99). However, there was a wide difference in the levels of Fpg-sensitive sites between the laboratory with the lowest (4.2% Tail DNA) and highest (74% Tail DNA) values in THP-1 cells after exposure to 4.5 mM KBrO3. In an attempt to assess sources of inter-laboratory variation in Fpg-sensitive sites, comet images from one experiment in each laboratory were forwarded to a central laboratory for visual scoring. There was high consistency between measurements of %Tail DNA values in each laboratory and the visual score of the same comets done in the central laboratory (r = 0.98, P < 0.001, linear regression). In conclusion, the results show that potassium bromate is a suitable positive comet assay control.

Comparison of comet-based approaches to assess base excision repair.

DNA repair plays an essential role in maintaining genomic stability, and can be assessed by various comet assay-based approaches, including the cellular repair assay and the in vitro repair assay. In the cellular repair assay, cells are challenged with a DNA-damaging compound and DNA damage removal over time is assessed. In the in vitro repair assay, an early step in the repair process is assessed as the ability of a cellular extract to recognize and incise damaged DNA in substrate nucleoids from cells treated with a DNA-damaging compound. Our direct comparison of both assays in eight cell lines and human peripheral blood lymphocytes indicated no significant relationship between these DNA repair assays (R2 = 0.084, P = 0.52). The DNA incision activity of test cells measured with the in vitro repair assay correlated with the background level of DNA damage in the untreated test cells (R2 = 0.621, P = 0.012). When extracts were prepared from cells exposed to DNA-damaging agents (10 mM KBrO3 or 1 µM Ro 19-8022 plus light), the incision activity was significantly increased, which is in line with the notion that base excision repair is inducible. The data presented suggest that the two assays do not measure the same endpoint of DNA repair and should be considered as complementary.

Visual comet scoring revisited: a guide to scoring comet assay slides and obtaining reliable results.

Measurement of DNA migration in the comet assay can be done by image analysis or visual scoring. The latter accounts for 20%-25% of the published comet assay results. Here we assess the intra- and inter-investigator variability in visual scoring of comets. We include three training sets of comet images, which can be used as reference for researchers who wish to use visual scoring of comets. Investigators in 11 different laboratories scored the comet images using a five-class scoring system. There is inter-investigator variation in the three training sets of comets (i.e. coefficient of variation (CV) = 9.7%, 19.8%, and 15.2% in training sets I-III, respectively). However, there is also a positive correlation of inter-investigator scoring in the three training sets (r = 0.60). Overall, 36% of the variation is attributed to inter-investigator variation and 64% stems from intra-investigator variation in scoring between comets (i.e. the comets in training sets I-III look slightly different and this gives rise to heterogeneity in scoring). Intra-investigator variation in scoring was also assessed by repeated analysis of the training sets by the same investigator. There was larger variation when the training sets were scored over a period of six months (CV = 5.9%-9.6%) as compared to 1 week (CV = 1.3%-6.1%). A subsequent study revealed a high inter-investigator variation when premade slides, prepared in a central laboratory, were stained and scored by investigators in different laboratories (CV = 105% and 18%-20% in premade slides with comets from unexposed and hydrogen peroxide-exposed cells, respectively). The results indicate that further standardization of visual scoring is desirable. Nevertheless, the analysis demonstrates that visual scoring is a reliable way of analysing DNA migration in comets.

Inter-laboratory variation in measurement of DNA damage by the alkaline comet assay in the hCOMET ring trial.

The comet assay is a simple and versatile method for measurement of DNA damage in eukaryotic cells. More specifically, the assay detects DNA migration from agarose gel-embedded nucleoids, which depends on assay conditions and the level of DNA damage. Certain steps in the comet assay procedure have substantial impact on the magnitude of DNA migration (e.g. electric potential and time of electrophoresis). Inter-laboratory variation in DNA migration levels occurs because there is no agreement on optimal assay conditions or suitable assay controls. The purpose of the hCOMET ring trial was to test potassium bromate (KBrO3) as a positive control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. To this end, participating laboratories used semi-standardized protocols for cell culture (i.e. cell culture, KBrO3 exposure, and cryopreservation of cells) and comet assay procedures, whereas the data acquisition was not standardized (i.e. staining of comets and image analysis). Segregation of the total variation into partial standard deviation (SD) in % Tail DNA units indicates the importance of cell culture procedures (SD = 10.9), comet assay procedures (SD = 12.3), staining (SD = 7.9) and image analysis (SD = 0.5) on the overall inter-laboratory variation of DNA migration (SD = 18.2). Future studies should assess sources of variation in each of these steps. On the positive side, the hCOMET ring trial demonstrates that KBrO3 is a robust positive control for the Fpg-modified comet assay. In conclusion, the hCOMET ring trial has demonstrated a high reproducibility of detecting genotoxic effects by the comet assay, but inter-laboratory variation of DNA migration levels is a concern.

Assay conditions for estimating differences in base excision repair activity with Fpg-modified comet assay.

DNA repair is an essential agent in cancer development, progression, prognosis, and response to therapy. We have adapted a cellular repair assay based on the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay to assess DNA repair kinetics. The removal of oxidized nucleobases over time (0-480 min) was analyzed in peripheral blood mononuclear cells (PBMCs) and 8 cell lines. DNA damage was induced by exposure to either Ro19-8022 plus visible light or potassium bromate (KBrO3). The initial amount of damage induced by Ro 19-8022 plus light varied between cell lines, and this was apparently associated with the rate of repair. However, the amount of DNA damage induced by KBrO3 varied less between cell types, so we used this agent to study the kinetics of DNA repair. We found an early phase of ca. 60 min with fast removal of Fpg-sensitive sites, followed by slower removal over the following 7 h. In conclusion, adjusting the initial damage at T0 to an equal level can be achieved by the use of KBrO3, which allows for accurate analysis of subsequent cellular DNA repair kinetics in the first hour after exposure.

Calibration of the comet assay using ionising radiation.

Several trials have attempted to identify sources of inter-laboratory variability in comet assay results, aiming at achieving more equal responses. Ionising radiation induces a defined level of DNA single-strand breaks (per dose/base pairs) and is used as a reference when comparing comet results but relies on accurately determined radiation doses. In this ring test we studied the significance of dose calibrations and comet assay protocol differences, with the object of identifying causes of variability and how to deal with them. Eight participating laboratories, using either x-ray or gamma radiation units, measured dose rates using alanine pellet dosimeters that were subsequently sent to a specialised laboratory for analysis. We found substantial deviations between calibrated and nominal (uncalibrated) dose rates, with up to 46% difference comparing highest and lowest values. Three additional dosimetry systems were employed in some laboratories: thermoluminescence detectors and two aqueous chemical dosimeters. Fricke's and Benzoic Acid dosimetry solutions gave reliable quantitative dose estimations using local equipment. Mononuclear cells from fresh human blood or mammalian cell lines were irradiated locally with calibrated (alanine) radiation doses and analysed for DNA damage using a standardised comet assay protocol and a lab-specific protocol. The dose response of eight laboratories, calculated against calibrated radiation doses, was linear with slope variance CV= 29% with the lab-specific protocol, reduced to CV= 16% with the standard protocol. Variation between laboratories indicate post-irradiation repair differences. Intra-laboratory variation was very low judging from the dose response of 8 donors (CV=4%). Electrophoresis conditions were different in the lab-specific protocols explaining some dose response variations which were reduced by systematic corrections for electrophoresis conditions. The study shows that comet assay data obtained in different laboratories can be compared quantitatively using calibrated radiation doses and that systematic corrections for electrophoresis conditions are useful.

Potassium bromate as positive assay control for the Fpg-modified comet assay.

The comet assay is a popular assay in biomonitoring studies. DNA strand breaks (or unspecific DNA lesions) are measured using the standard comet assay. Oxidative stress-generated DNA lesions can be measured by employing DNA repair enzymes to recognise oxidatively damaged DNA. Unfortunately, there has been a tendency to fail to report results from assay controls (or maybe even not to employ assay controls). We believe this might have been due to uncertainty as to what really constitutes a positive control. It should go without saying that a biomonitoring study cannot have a positive control group as it is unethical to expose healthy humans to DNA damaging (and thus potentially carcinogenic) agents. However, it is possible to include assay controls in the analysis (here meant as a cryopreserved sample of cells i.e. included in each experiment as a reference sample). In the present report we tested potassium bromate (KBrO3) as a positive comet assay control for the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay. Ten laboratories used the same procedure for treatment of monocytic THP-1 cells with KBrO3 (0.5, 1.5 and 4.5 mM for 1 h at 37°C) and subsequent cryopreservation. Results from one laboratory were excluded in the statistical analysis because of technical issues in the Fpg-modified comet assay. All other laboratories found a concentration-response relationship in cryopreserved samples (regression coefficients from 0.80 to 0.98), although with different slopes ranging from 1.25 to 11.9 Fpg-sensitive sites (%DNA in tail) per 1 mM KBrO3. Our results demonstrate that KBrO3 is a suitable positive comet assay control.

Isolation of leukocytes from frozen buffy coat for comet assay analysis of DNA damage.

Frozen buffy coat fractions are often stored in human biomonitoring trials but their use for biomarker purposes has been limited. The purpose of the current study was to study whether frozen buffy coats can be used to monitor DNA damage levels. EDTA blood samples were provided from 9 healthy, non-smoking female volunteers, aged 26-48. Pre-existing DNA damage (strand breaks and oxidised purines) was measured with the comet assay in thawed resuspended buffy coat samples and washed leukocytes from these buffy coats, as well as resistance to DNA damage induced exogenously by H2O2 in the latter, and compared with damage measured in peripheral blood mononuclear cells isolated from fresh blood using percoll gradient centrifugation. Basal DNA damage levels (strand breaks) were significantly higher in the leukocytes isolated from frozen buffy coats in the untreated samples compared with peripheral blood mononuclear cells. However, the levels of strand breaks were still low (<4% tail DNA), indicating that little damage is caused by freezing or processing. Base oxidation was significantly higher in isolated buffy coat leukocytes than in peripheral blood mononuclear cells from fresh blood, but showed a good correlation (r = 0.67) between the two cell types. The correlation for strand breaks was stronger (r = 0.85). H2O2 induced DNA breaks in the cells both from fresh blood and buffy coats. The results indicate that buffy coat samples stored from cohort studies might be usefully analysed for DNA damage in retrospective epidemiological investigations. However, caution should be exercised when comparing the absolute levels of DNA damage in buffy coat leukocytes and peripheral blood mononuclear cells.

Prevention of Deficit in Neuropsychiatric Disorders through Monitoring of Arsenic and Its Derivatives as Well as Through Bioinformatics and Cheminformatics.

Neuropsychiatric disorders are induced by various risk factors, including direct exposure to environmental chemicals. Arsenic exposure induces neurodegeneration and severe psychiatric disorders, but the molecular mechanisms by which brain damage is induced are not yet elucidated. Our aim is to better understand the molecular mechanisms of arsenic toxicity in the brain and to elucidate possible ways to prevent arsenic neurotoxicity, by reviewing significant experimental, bioinformatics, and cheminformatics studies. Brain damage induced by arsenic exposure is discussed taking in account: the correlation between neuropsychiatric disorders and the presence of arsenic and its derivatives in the brain; possible molecular mechanisms by which arsenic induces disturbances of cognitive and behavioral human functions; and arsenic influence during psychiatric treatments. Additionally, we present bioinformatics and cheminformatics tools used for studying brain toxicity of arsenic and its derivatives, new nanoparticles used as arsenic delivery systems into the human body, and experimental ways to prevent arsenic contamination by its removal from water. The main aim of the present paper is to correlate bioinformatics, cheminformatics, and experimental information on the molecular mechanism of cerebral damage induced by exposure to arsenic, and to elucidate more efficient methods used to reduce its toxicity in real groundwater.

Methamphetamine ("crystal meth") causes induction of DNA damage and chromosomal aberrations in human derived cells.

Methamphetamine (METH) is a widely consumed psychostimulant drug; its acute toxic effects in brain and liver are well known, furthermore, there is some evidence in regard to its DNA damaging properties in humans. Therefore, we studied the impact of the drug on genomic stability in human derived hepatoma (HepG2) cells, which reflect the activation/detoxification of drugs better than other cell lines. Furthermore, experiments with human buccal derived cells (TR146) were conducted as the drug is consumed orally. Induction of DNA damage in both cell types with doses reflecting the exposure in abusers was found in single cell gel electrophoresis (SCGE) assays (which detect single and double strand breaks as well as apurinic sites). Furthermore, induction of micronuclei (formed as a consequence of structural and numerical chromosomal aberrations) and formation of nuclear buds resulting from gene amplifications was detected. Additional experiments with lesion-specific enzymes showed that the drug causes oxidation of purines and pyrimidines, indicating that its genotoxic effects may be due to oxidation of the DNA. Our findings support the assumption that the drug may cause adverse health effects (such as cancer and infertility) in long-term users which are causally related to DNA damage.

Consumption of a dark roast coffee blend reduces DNA damage in humans: results from a 4-week randomised controlled study.

PURPOSE: To determine the DNA protective effects of a standard coffee beverage in comparison to water consumption. METHODS: The single-blind, randomised controlled study with parallel design included healthy women (n = 50) and men (n = 50) recruited from the general Central European population. The subjects were randomised in a coffee and a control group, with stratification for sex and body mass index. The study comprised two periods of 4 weeks: a preconditioning period, with daily consumption of at least 500 ml water but no coffee, nor tea, nor any other caffeine-containing product. During the subsequent intervention period the coffee group consumed 500 ml of freshly brewed dark roast coffee blend per day, the control group consumed water instead. On the last day of each period, blood was drawn and analysed by comet assay (single-cell gel electrophoresis) to assess the level of DNA damage (strand breakage). RESULTS: At the end of the intervention period the mean level of DNA strand breaks in the coffee group has decreased in comparison to the control group [difference in means 0.23% TI (tail intensity), p = 0.028]. The mean change from baseline (delta value) was - 23% in the coffee group (p = 0.0012). Effects of coffee intake were similar for men and women. During intervention, neither group showed any significant change in body weight or calorie intake. CONCLUSIONS: Our results indicate that regular consumption of a dark roast coffee blend has a beneficial protective effect on human DNA integrity in both, men and women.

Characterization of the proteome and lipidome profiles of human lung cells after low dose and chronic exposure to multiwalled carbon nanotubes.

The effects of long-term chronic exposure of human lung cells to multi-walled carbon nanotubes (MWCNT) and their impact upon cellular proteins and lipids were investigated. Since the lung is the major target organ, an in vitro normal bronchial epithelial cell line model was used. Additionally, to better mimic exposure to manufactured nanomaterials at occupational settings, cells were continuously exposed to two non-toxic and low doses of a MWCNT for 13-weeks. MWCNT-treatment increased ROS levels in cells without increasing oxidative DNA damage and resulted in differential expression of multiple anti- and pro-apoptotic proteins. The proteomic analysis of the MWCNT-exposed cells showed that among more than 5000 identified proteins; more than 200 were differentially expressed in the treated cells. Functional analyses revealed association of these differentially regulated proteins to cellular processes such as cell death and survival, cellular assembly, and organization. Similarly, shotgun lipidomic profiling revealed accumulation of multiple lipid classes. Our results indicate that long-term MWCNT-exposure of human normal lung cells at occupationally relevant low-doses may alter both the proteome and the lipidome profiles of the target epithelial cells in the lung.

Coffee and oxidative stress: a human intervention study.

PURPOSE: Coffee is known to contain phytochemicals with antioxidant potential. The aim of this study was to investigate possible antioxidant effects of coffee in healthy human volunteers. METHODS: A placebo-controlled intervention trial was carried out on 160 healthy human subjects, randomised into three groups, receiving 3 or 5 cups of study coffee or water per day, for 8 weeks. Blood samples were taken before, during, and after the intervention. Serum was used for analysis of blood lipids and standard clinical chemistry analytes. Peripheral blood mononuclear cells were isolated, and DNA damage (strand breaks and oxidised bases) was measured with the comet assay. The lipid oxidation product isoprostane 8-iso-PGF2α was assayed in urine samples by LC-MS/MS. RESULTS: There was no significant effect of coffee consumption on the markers of oxidation of DNA and lipids. Creatinine (in serum) increased by a few per cent in all groups, and the liver enzyme γ-glutamyl transaminase was significantly elevated in serum in the 5 cups/day group. Other clinical markers (including glucose and insulin), cholesterol, triacylglycerides, and inflammatory markers were unchanged. There was no effect of coffee on blood pressure. CONCLUSION: In a carefully controlled clinical trial with healthy subjects, up to 5 cups of coffee per day had no detectable effect, either beneficial or harmful, on human health.

Cellular responses of human astrocytoma cells to dust from the Acheson process: An in vitro study.

Silicon carbide (SiC) is largely used in various products such as diesel particulate filters and solar panels. It is produced through the Acheson process where aerosolized fractions of SiC and other by-products are generated in the work environment and may potentially affect the workers' health. In this study, dust was collected directly on a filter in a furnace hall over a time period of 24h. The collected dust was characterized by scanning electron microscopy and found to contain a high content of graphite particles, and carbon and silicon containing particles. Only 6% was classified as SiC, whereof only 10% had a fibrous structure. To study effects of exposure beyond the respiratory system, neurotoxic effects on human astrocytic cells, were investigated. Both low, occupationally relevant, and high doses from 9E-6µg/cm2 up to 4.5µg/cm2 were used, respectively. Cytotoxicity assay indicated no effects of low doses but an effect of the higher doses after 24h. Furthermore, investigation of intracellular reactive oxygen species (ROS) indicated no effects with low doses, whereas a higher dose of 0.9µg/cm2 induced a significant increase in ROS and DNA damage. In summary, low doses of dust from the Acheson process may exert no or little toxic effects, at least experimentally in the laboratory on human astrocytes. However, higher doses have implications and are likely a result of the complex composition of the dust.